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Site-saturation mutagenesis by overlap extension PCR

Authors :
Elsie M, Williams
Janine N, Copp
David F, Ackerley
Source :
Methods in molecular biology (Clifton, N.J.). 1179
Publication Year :
2014

Abstract

Site-saturation mutagenesis is a proven strategy for generating high-quality variant gene libraries of a defined size. Variation is introduced via incorporation of degenerate base combinations at specific codon locations, giving rise to a precise series of amino acid substitutions in the encoded protein. Here we describe a simple and efficient overlap PCR protocol for the introduction of degenerate bases at either single or multiple codon locations. The resulting libraries can then be directly screened for improved protein function as either an independent directed evolution study or an adjunct to random mutagenesis strategies (such as error-prone PCR) that are, in isolation, unlikely to access the full repertoire of possible amino acid substitutions at any given position.

Details

ISSN :
19406029
Volume :
1179
Database :
OpenAIRE
Journal :
Methods in molecular biology (Clifton, N.J.)
Accession number :
edsair.pmid..........9068ec6b34a9bec29e154f350dbcf545